Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms.

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any ß subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

Ultrafast, Ultrasensitive Detection and Imaging of Single Cardiac Troponin-T Molecules.

The fluorescence-based methods of single-molecule optical detection have opened up unprecedented possibilities for imaging, monitoring, and sensing at a single-molecule level. However, single-molecule detection methods are very slow, making them practically inapplicable. In this paper, we show how to overcome this key limitation using the expanded laser spot, laser excitation in a nonfluorescent spectral window of biomolecules, and more binding fluorescent molecules on a biomolecule that increases the detection volume and the number of collected photons. We demonstrate advantages of the developed approach unreachable by any other technique using detection of single cardiac troponin-T molecules: (i) 1000-fold faster than by known approaches, (ii) real-time imaging of single troponin-T molecules dissolved in human blood serum, (iii) measurement of troponin-T concentration with a clinically important sensitivity of about 1 pg/mL. The developed approach can be used for ultrafast, ultrasensitive detection, monitoring, and real-time imaging of other biomolecules as well as of larger objects including pathogenic viruses and bacteria.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

Neurotoxins from snake venoms and α-conotoxin ImI inhibit functionally active ionotropic γ-aminobutyric acid (GABA) receptors.

Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1ß3γ2 receptor; and at 10 µm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1ß2γ2 ≈ α2ß2γ2 > α5ß2γ2 > α2ß3γ2 and α1ß3δ GABAARs. The α1ß3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the ß/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors.